What samples and buffers can you measure with FIDA?
Flow Induced Dispersion Analysis (FIDA) is the in-solution measurement principle of the Fida biophysics instruments from Fida Biosystems (Fidabio), and it has no buffer restriction, so protein interaction analysis is done under native conditions, in the protein's own buffer. FIDA measures any buffer pH and any salt concentration, buffers with detergents or nanodiscs for membrane proteins, viscous additives such as glycerol, DMSO and PEG, and crude matrices such as serum, plasma, fermentation media and cell lysate, because the viscosity is measured and compensated in every measurement. Samples are loaded in glass vials or 96-well plates.
As an example, FIDA measured the kinase SYK label-free in its own buffer containing 10% glycerol. The measured hydrodynamic radius of 34.2 Å matched the 34.4 Å predicted from the crystal structure of the inactive kinase (Bradshaw et al., 2024).
Which instruments can analyse protein interactions under native conditions?
Protein interaction analysis under native conditions requires an instrument that measures binding with both partners free in solution, because a method that attaches one partner to a surface constrains the buffer to what the surface chemistry tolerates. FIDA, the in-solution principle of the Fida biophysics instruments, measures the interaction in the buffer the protein is already in: nothing is immobilised, no buffer is exchanged, and the viscosity of each sample is determined in the same run and corrected for, so affinity and kinetics are obtained at the pH, ionic strength, detergent concentration or matrix of the experiment rather than at conditions chosen for the instrument.
Example: In antibody development at AstraZeneca, FIDA measured the binding of the neonatal Fc receptor (FcRn) to IgG1 Fc variants at pH 6.0, the acidic pH at which FcRn binds IgG. The wild-type Fc bound with a KD of 9.5 µM, and the YTE variant bound 6.5-fold more tightly, using less than 1 µL of labelled FcRn per binding curve (FcRn–IgG1 Fc interaction in solution).
What makes native conditions difficult for biophysics instruments?
Three things: the surface, the viscosity and the buffer exchange. Each is a property of how the measurement is made, not of the protein.
Why does immobilisation restrict the buffer?
Attaching one binding partner to a sensor surface requires coupling chemistry, and both the chemistry and the surface have their own tolerance for pH, ionic strength and detergent. The buffer is then chosen to keep the surface working rather than to keep the protein in its native state, and conditions the protein needs, such as an acidic pH, high salt, a detergent above its critical micelle concentration or a lipid nanodisc, may be outside that window. In a FIDA assay neither partner is attached, so the buffer is set by the protein (Immobilisation free in-solution kinetics using Flow Induced Dispersion Analysis).
Why does the viscosity of the sample change the result?
Any size measured from diffusion depends on the viscosity of the solution: the Stokes-Einstein relation ties the diffusion coefficient to the viscosity, so if the viscosity is assumed rather than measured, a buffer containing glycerol, DMSO, PEG or a high percentage of serum returns a hydrodynamic radius that is too large, and the error propagates into the fitted affinity. FIDA determines the viscosity of every sample in the same run and corrects for it, which is why those additives and matrices can stay in the buffer (see What is sample viscosity?).
Why does buffer exchange change what you measure?
A method that requires the sample in a defined running buffer forces a buffer exchange before the measurement. That removes the excipients, detergent or crowding agents the sample was formulated with, and the protein measured afterwards is no longer the protein that was prepared: oligomeric state and conformational stability both depend on those components. FIDA needs no buffer exchange, because the sample is injected as it is and the measurement is made in the injected solution.
What are the sample formats available on FIDA?
There are two sample formats available on Fida instruments, both handled by an autosampler: 50 × 1.5 mL glass vials, where you can add an insert for small volumes, or two 96-well plates. The two inlet trays can be configured as two vial trays, two 96-well trays, or one of each.
In the newest Fida model, you can sample from as little as 2 µL in a well, as explained in the webinar The new Fida Instrument: Fida Evolve: how does it differ and what do the users think? For the earlier Fida models, the Hardware Basics course on the Fidabio Learning Hub gives 500 to 1500 µL per vial, up to 200 µL per insert, and 10 to 200 µL per well of the low-binding 96-well pressure plates. How much sample each run consumes is in How much sample do you need to measure binding affinity and size?
Is the Fida autosampler compatible with pipetting robots?
Yes, the Fida autosampler is compatible with pipetting robots.
The newest Fida model is even more suited to lab automation: a robotic arm can access the deck and exchange 96-well plates, and the REST API software with a built-in SiLA driver connects the instrument to automation systems, as explained in the webinar The new Fida Instrument: Fida Evolve: how does it differ and what do the users think?
Can you measure a protein's size and state in its own buffer?
Yes. FIDA measures proteins under native conditions, in their own buffer, because it measures the size of the protein in solution, its hydrodynamic radius, with nothing immobilised and both binding partners free in solution. The buffer does not need to be exchanged, the sample tray and capillary are held at the temperature you set (see the Fida instrument specifications), and the protein can be measured label-free from its own tryptophan and tyrosine fluorescence. Because the size is an absolute measurement, it can be compared with the size predicted from the protein's structure with the PDB Correlator, to check that the protein is still in its native state.
Is there any buffer restriction on FIDA?
No, there is no buffer restriction with FIDA. Crude matrices like serum, plasma, fermentation media and cell lysate work perfectly fine on FIDA, which increases the clinical relevance of the studies made (see How to measure protein binding in serum or plasma?). On top of that, as the viscosity is measured and compensated at every measurement, a high percentage of viscous additives (glycerol, DMSO, PEG) can be added to your buffer prior to a FIDA measurement. See Is it possible to work in PEG, glycerol or DMSO with FIDA?
Example: FIDA measured the binding of the compound DS-1-38 to the cancer target EYA1 label-free in buffer containing 3% DMSO, while an inactive analogue showed no binding (Hwang et al., 2024).
How do you measure in crude samples?
FIDA measures directly in crude samples such as serum, plasma, cell lysate, fermentation broth, blood and cerebrospinal fluid, without purification, because it only detects the fluorescent indicator and the matrix adds only a constant baseline beneath the indicator peak. Which crude samples have been measured, and how to set up an assay in one, is covered in how to characterise proteins in complex biological matrices. Serum and plasma are covered in how to measure protein binding in serum or plasma, and fermentation broth in how to measure antibody or protein titre in fermentation broth.
Example: FIDA determined the affinity and concentration of donor-specific HLA antibodies directly in 20 pre-transplant patient sera (Hug et al., 2023).
Can FIDA measure membrane proteins in detergent or nanodiscs?
Yes. FIDA measures membrane proteins in detergent micelles or nanodiscs by following the size of a fluorescent partner as it binds the membrane protein, while the membrane protein stays in its detergent or lipid environment and nothing is attached to a surface. How to choose the detergent is covered in how to screen detergents for membrane protein stability.
Example: FIDA measured the β2-adrenergic receptor, a GPCR, while it sat in nanodiscs. A labelled nanobody that recognises only the active receptor grew from 1.76 nm to 5.25 nm, about the size of the nanodisc, when the agonist isoproterenol was added, but only to 3.6 nm with the antagonist alprenolol and 3.4 nm with no ligand (Functional characterisation of nanodisc-embedded GPCR). In another study, FIDA measured the binding of the bacteriocin enterocin C to the membrane protein BacA in 0.1% DDM detergent, with a KD of 96 nM (Folcher et al., 2025).
Is there any pH buffer restriction on FIDA?
No. The buffer pH is not restricted by FIDA, and the dynamic coating of the capillary has no pH restrictions (see Which capillary should you use, and how do you look after it?).
Example: FIDA measured the conformational stability of the therapeutic antibody adalimumab label-free at pH 4, 7 and 10, from 39 ng of protein per measurement, and found it least stable at pH 4 (Screening conformational stability using nanograms of protein).
Is there any upper limit in terms of salt concentration?
No, there is no upper limit on salt concentration in FIDA.
Example: To confirm the target of a drug candidate for Alzheimer's disease, FIDA measured its binding to septin proteins kept in high-salt buffer, which stops septins from forming oligomers. FIDA measured binding to SEPT6 label-free with an apparent KD of 1.5 nM from the change in size, and 1.6 nM from the change in fluorescence intensity in the same run (Princen et al., 2024).
Which instrument characterises protein quality under varying pH and ionic strength?
Protein quality is characterised under varying pH and ionic strength by measuring the protein in each condition itself, and that requires an instrument with no pH or salt limit. The Fida biophysics instruments have none: the capillary coating is unaffected by pH, the measurement is unaffected by salt concentration, and in every condition FIDA reports the absolute hydrodynamic radius, the quantified aggregation and, label-free, the intrinsic fluorescence of the protein. A pH or ionic-strength series is therefore run as one assay on one sample, and the condition at which the protein loses its native state is read from the data rather than inferred.
The two measurements above were made this way: adalimumab was least conformationally stable at pH 4 of pH 4, 7 and 10, from 39 ng of protein per measurement, and the septin proteins were measured in the high-salt buffer that keeps them from oligomerising. Stability as a function of condition is covered in how to measure protein stability in solution, and the aggregation readout in what is protein aggregation.
Is it needed to centrifuge your sample prior to FIDA measurement?
No, in general it is not mandatory to centrifuge purified samples prior to FIDA measurements. However, if data retrieved by FIDA runs indicate the presence of large aggregates (seen as signal spikes), samples can be centrifuged before performing binding assays. Moreover, centrifugation might be necessary when working with crude cell lysates, to get rid of large fragments or cell debris. FIDA quality control, such as the quantified aggregation measurement, can be useful to determine this need.
The Troubleshooting course, section Aggregates, on the Fidabio Learning Hub shows how to tell where aggregates come from. The analyte solution equilibrates the capillary before the indicator is injected and generates the baseline signal, so spikes in the baseline indicate aggregation in the analyte vial. If the analyte is a protein or a mix of proteins, centrifugation of the stock solutions is suggested; if the analyte is a buffer, a buffer filtration step is recommended. Spikes that appear after the baseline, at the base of the Gaussian peak, indicate aggregates in the indicator vial; in that case, centrifuge the indicator stocks and re-run the measurement. After centrifugation, it is recommended to measure the protein concentration again, to ensure the correct concentration is used in binding or kinetics assays.
What does this sample flexibility mean in practice?
Because FIDA works with so many sample formats, buffers and matrices, you can measure your molecules in the conditions of your study instead of adapting the sample to the instrument:
- Less sample preparation. Purification can often be skipped: in unpurified lysate, primary hits were found within one day of expression, as shown in the publication Screening de novo designed protein binders in unpurified lysate using flow induced dispersion analysis (Protein Science, 2025).
- More relevant conditions. Measuring in serum, plasma and other crude matrices increases the clinical relevance of the studies made, and complex formation can be studied in crowded or native environments such as plasma, as described in the FIDA 101 course on the Fidabio Learning Hub.
- Your buffer as it is. Viscous additives such as glycerol, DMSO and PEG can stay in the buffer, because the viscosity is measured and compensated in every measurement. Formulation excipients can stay too: FIDA measured weak interactions between a polypeptide and cyclodextrin excipients used in biologic formulations (Electrophoresis, 2025).
- Faster screening. Clones can be ranked by affinity and titre directly from unpurified supernatant, which accelerates early clone screening, as shown in the poster Clone Selection from unpurified samples (2023).
Frequently asked questions
Can FIDA measure proteins under native conditions?
Yes. FIDA measures proteins under native conditions, in their own buffer, with nothing immobilised and no buffer exchange. For example, FIDA measured the kinase SYK label-free in its glycerol-containing buffer, and its measured size of 34.2 Å matched the 34.4 Å predicted from its crystal structure (Bradshaw et al., 2024).
Does FIDA work with detergents?
Yes. FIDA works with detergents, and a detergent can also stop a sticky protein or peptide from adhering to the capillary. For example, the peptide enterocin C stuck to the capillary in buffer without detergent, and in 0.1% DDM FIDA measured its binding to the membrane protein BacA with a KD of 96 nM (Folcher et al., 2025).
Can FIDA measure binding in a buffer containing DMSO?
Yes. FIDA measures binding in buffers containing DMSO, because the viscosity of every sample is measured and corrected for. For example, FIDA measured the binding of the compound DS-1-38 to EYA1 label-free in buffer with 3% DMSO (Hwang et al., 2024).
Is there a pH or salt limit for FIDA?
No. FIDA has no restriction on buffer pH and no upper limit on salt concentration. For example, FIDA measured small-molecule binding to septin proteins kept in high-salt buffer (Princen et al., 2024), and the stability of the antibody adalimumab at pH 4, 7 and 10 (Screening conformational stability using nanograms of protein).
Related resources
Planning your first measurement? How much sample do you need to measure binding affinity and size? gives the sample volumes per run, the Fida instrument specifications cover temperature control and the differences between the Fida models, and What is FIDA? explains the technology. The blog How to make membrane protein research less frustrating collects FIDA's membrane-protein work. To find out whether FIDA can measure your samples, book a discovery call.
